Cell culture medium for culturing organoid, culture method, and organoid

ABSTRACT

A cell culture medium for culturing organoid containing at least two types of components selected from the group consisting of insulin-like growth factor 1 (IGH1), fibroblast growth factor 2 (FGF2) and epiregulin (EREG), and at least one type of component among the following components i (to III); i) Wnt agonist, ii) bone morphogenetic protein (BMP) inhibitor, and iii) transforming growth factor-β (TGIF-β) inhibitor.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 16/301,717 filed Sep. 11, 2019, which is a U.S.National Phase Application under 35 U.S.C. § 371 of International PatentApplication No. PCT/JP2017/017681, filed May 10, 2017, which claimspriority of Japanese Patent Application No. 2016-099995, filed May 18,2016. The entire contents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a cell culture medium for culturing anorganoid, a culture method and an organoid.

BACKGROUND

The intestinal tract is the organ that has the largest surface area incontact with the outside world in the body, and has functions that areindispensable for maintaining life such as digestion and absorption. Themajority of intestinal tract functions are fulfilled by intestinalepithelium that covers the inner layer thereof. Intestinal epithelium iscomposed of two components consisting of cilia comprised of three typesof differentiated cells (mucus-producing cells, absorptive epithelialcells and endocrine cells) and crypts mainly comprised ofundifferentiated germ cells. Paneth cells that produce antimicrobialpeptides are present at the base of crypts of the small intestine.Recently, Lgr5-positive cells (also referred to as crypt base columnar(CBC) cells) have been determined by molecular genetics-based celllineage analysis to be intestinal epithelial stem cells. AlthoughLgr5-positive intestinal epithelial stem cells produce progenitor cellsreferred to as transit amplifying cells, these progenitor cells do nothave a permanent self-replicating ability, but rather differentiationability is limited to one to three cell lines. Transit amplifying cellsdifferentiate together with two to four cycles of cell division incrypts and achieve terminal differentiation in cilia. Thesedifferentiated cells detach at the tips of cilia and die due toapoptosis. Intestinal epithelium is tissue that has a fast metabolismand migrates from crypt stem cells to the tips of cilia in four to fivedays. Differing from other differentiated cells, Paneth cells migrate tothe bottoms of crypts accompanying differentiation and have a long celllife of two months.

The self-replication mechanism of intestinal epithelial stem cells isknown to be controlled by a Wnt signal and bone morphogenetic protein(BMP) signal based on results in several genetically modified mice.Knockout mice specific for intestinal epithelium expressing Wnt signalinhibitory molecules in the form of adenomatous polyposis coli (APC)exhibit hyperproliferation and adenoma formation. In addition, ectopiccrypt formation has been observed in mice demonstrating overexpressionof BMP inhibitory protein in the form noggin in intestinal epithelialcells, suggesting that the BMP signal acts to inhibit intestinalepithelial stem cells. In actuality, the Wnt signal is highly active atthe bases of crypts and a gradient has been observed by which thatactivity becomes lower towards the lumen. On the other hand, the BMPsignal is known to exhibit a gradient opposite that from the Wnt signal.

In addition, long-term culturing of intestinal epithelial cells has longbeen impossible. This is thought to be due to the growth factorsrequired for maintaining intestinal epithelial stem cells being unknown.In recent years, long-term maintenance of intestinal epithelial stemcells has been successful by adhering intestinal epithelial stem cellsto extracellular matrix and culturing in the presence of cell culturemedium containing basal medium for animal or human cells to which hasbeen added BMP inhibitor, mitogenic growth factor and Wnt agonist (see,for example, Japanese Patent No. 5458112).

SUMMARY Problems to be Solved by the Invention

The cell culture media used in the culture method described in JapanesePatent No. 5458112 had the problem of a decrease in expression ofepidermal growth factor receptor (EGFR) caused by stimulation ofepidermal growth factor (EGF). In contrast, the addition of p38inhibitor made it possible to maintain expression of EGFR. However,media and culture methods incorporating p38 inhibitor may causeinhibition of differentiation or cell death, thereby preventingculturing of some human tissue and tumor tissue.

In order to solve the aforementioned problems, the present inventionprovides a cell culture medium for culturing organoid enabling long-termculturing of epithelial stem cells, epithelial cells or epithelial tumorcells derived from mammals, including humans, tissue at least containingany one of these cells, or tissue unable to be cultured in the priorart.

In addition, the present invention provides a cultured human organoidable to be produced in the absence of serum. In addition, the presentinvention provides a cultured human organoid produced from tissue inwhich the production thereof was previously not possible.

As a result of conducting extensive studies to solve the aforementionedproblems, the inventors of the present invention discovered thecomposition of a cell culture medium for culturing organoid that enableslong-term culturing of epithelial stem cells, epithelial cells orepithelial tumor cells derived from mammals, including humans, tissue atleast containing any one of these cells, or tissue unable to be culturedin the prior art. In addition, the inventors of the present inventionfound that an organoid can be formed by culturing epithelial stem cells,epithelial cells or epithelial tumor cells derived from mammals,including humans, tissue at least containing any one of these cells, ortissue unable to be cultured in the prior art, under hypoxic condition.

Namely, the present invention includes the aspects indicated below.

The cell culture medium for culturing organoid according to a firstaspect of the present invention contains at least two types ofcomponents selected from the group consisting of insulin-like growthfactor 1 (IGF1), fibroblast growth factor 2 (FGF2) and epiregulin(EREG), and at least one type of component among the followingcomponents i) to iii):

-   -   i) Wnt agonist,    -   ii) bone morphogenetic protein (BMP) inhibitor, and    -   iii) transforming growth factor-β (TGF-β) inhibitor.

The cell culture medium for culturing organoid according to theaforementioned first aspect may substantially not contain EGF and p38inhibitor.

The cell culture method for culturing organoid according to theaforementioned first aspect may also contain IGF1 and FGF2.

The Wnt agonist may be at least one type selected from the groupconsisting of Wnt protein, R-spondin and GSK-3β inhibitor.

The Wnt protein may form a complex with afamin, which is a stabilizingsubstance thereof.

The Wnt agonist may be Wnt protein and R-spondin.

The cell culture medium for culturing organoid described in any one ofclaims 4 to 6, wherein the Wnt protein is at least one type selectedfrom the group consisting of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4,Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a,Wnt10b, Wnt11 and Wnt16.

The R-spondin may be at least one type selected from the groupconsisting of R-spondin 1, R-spondin 2, R-spondin 3 and R-spondin 4.

The BMP inhibitor may be at least one type selected from the groupconsisting of noggin, gremlin, chordin, chordin-like protein containinga chordin domain, follistatin, follistatin-related protein containing afollistatin domain, DAN, DAN-like protein containing a DAN cysteine knotdomain, sclerostin/SOST and α-2 macroglobulin.

The BMP inhibitor may be noggin.

The TGF-β inhibitor may be at least one type selected from the groupconsisting of A83-01, SB-431542, SB-505124, SB-525334, SD-208, LY-36494and SJN-2511.

The TGF-β inhibitor may be A83-01.

The culture method according to a second aspect of the present inventionis a method for culturing epithelial stem cells, epithelial cells,epithelial tumor cells or tissue at least containing any one of thesecells; provided with: an extracellular matrix preparation step forpreparing an extracellular matrix, an adhesion step for adheringepithelial stem cells, epithelial cells, epithelial tumor cells ortissue at least containing any one of these cells onto the extracellularmatrix, and an organoid formation step for forming an organoid by addingthe cell culture medium for culturing organoid according to theaforementioned first aspect and culturing the epithelial stem cells, theepithelial cells, the epithelial tumor cells or tissue at leastcontaining any one of these cells, after the cell adhesion step.

In the organoid formation step, an organoid may be formed by culturingthe epithelial stem cells, the epithelial cells, the epithelial tumorcells or tissue at least containing any one of these cells under hypoxiccondition at an oxygen concentration of 15% to 0.1%.

The organoid containing differentiated cells according to a third aspectof the present invention is obtained by the method according to theaforementioned second aspect.

The organoid containing cells differentiated cells according to theaforementioned third aspect may be for regenerative medicine.

The culture method according to a fourth aspect of the present inventionis a method for culturing the epithelial stem cells, the epithelialcells, the epithelial tumor cells or tissue at least containing any oneof these cells; provided with: an extracellular matrix preparation stepfor preparing an extracellular matrix, an adhesion step for adheringepithelial stem cells, epithelial cells, epithelial tumor cells ortissue at least containing any one of these cells onto the extracellularmatrix, and an organoid formation step for forming an organoid by addingthe cell culture medium for culturing organoid and culturing theepithelial stem cells, the epithelial cells, the epithelial tumor cellsor tissue at least containing any one of these cells under hypoxiccondition at an oxygen concentration of 15% to 0.1%, after the celladhesion step; wherein, the cell culture medium for culturing organoidcontains at least one type selected from the group consisting of Wntagonist, mitogenic growth factor, BMP inhibitor, TGF-β inhibitor and p38inhibitor.

The Wnt agonist may be at least one type selected from the groupconsisting of Wnt protein, R-spondin and GSK-3β inhibitor.

The Wnt protein may form a complex with afamin, which is a stabilizingsubstance thereof.

The Wnt agonist may be Wnt protein and R-spondin.

The Wnt protein may be at least one type selected from the groupconsisting of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6,Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11 andWnt16.

The R-spondin may be at least one type selected from the groupconsisting of R-spondin 1, R-spondin 2, R-spondin 3 and R-spondin 4.

The BMP inhibitor may be at least one type selected from the groupconsisting of noggin, gremlin, chordin, chordin-like protein containinga chordin domain, follistatin, follistatin-related protein containing afollistatin domain, DAN, DAN-like protein containing a DAN cysteine knotdomain, sclerostin/SOST and α-2 macroglobulin.

The BMP inhibitor may be noggin.

The TGF-β inhibitor may be at least one type selected from the groupconsisting of A83-01, SB-431542, SB-505124, SB-525334, SD-208, LY-36494and SJN-2511.

The TGF-β inhibitor may be A83-01.

The organoid containing differentiated cells according to a fifth aspectof the present invention is obtained by the culture method according tothe aforementioned fourth aspect.

The organic containing differentiated cells according to theaforementioned fifth aspect may be for regenerative medicine.

According to the cell culture method for culturing organoid of theaforementioned aspects, epithelial stem cells, epithelial cells orepithelial tumor cells derived from mammals, including humans, tissue atleast containing any one of these cells, or tissue unable to be culturedin the prior art, can be cultured for a long period of time. Inaddition, an organoid can be formed with high efficiency from at leastany one of the aforementioned cells and aforementioned tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts images showing cultured organoids of human epithelialtumor cells on day 7 from the start of primary culturing (passage 0) ineach of the media of Example 1. Here, W represents Wn-3A, N representsnoggin, R represents R-spondin 1 and A represents A83-01.

FIG. 2 is a graph quantifying the area in wells occupied by organoids ofhuman epithelial tumor cells on day 7 from the start of primaryculturing (passage 0) in each of the media of Example 1. Theabbreviations have the same meanings as in FIG. 1 .

FIG. 3 depicts images indicating cultured organoids of human epithelialtumor cells on day 7 from the start of primary culturing (passage 0) ateach oxygen concentration in Example 2.

FIG. 4 is a diagram indicating optimal culturing conditions fororganoids established from 55 types of colon tumors.

FIG. 5A depicts images of cultured organoids of human intestinal stemcells on day 7 from the start of primary culturing (passage 0) in eachof the media of Example 3.

FIG. 5B is a graph quantifying the area in wells occupied by organoidsof human intestinal stem cells on day 7 from the start of primaryculturing (passage 0) in each of the media of Example 1.

FIG. 6 depicts images indicating organoids of human epithelial tumorcells six days after a third round of subculturing (passage 3) (totalnumber of days cultured: 26), six days after the start of a fourth roundof subculturing (passage 4) (total number of days cultured: 33), and sixdays after the start of a sixth round of subculturing (passage 6) (totalnumber of days cultured; 47).

FIG. 7 is a table indicating the results of having evaluated organoidformation efficiency in cell culture medium for culturing organoid ofthe present embodiment having different compositions.

DETAILED DESCRIPTION

<<Cell Culture Medium for Culturing Organoid>>

The cell culture medium for culturing organoid according a firstembodiment of the present invention contains at least two types ofcomponents selected from the group consisting of IGF1, FGF2 and EREG,and at least one type of component among the following components i) toiii):

-   -   i) Wnt agonist,    -   ii) bone morphogenetic protein (BMP) inhibitor, and    -   iii) transforming growth factor-β (TGF-β) inhibitor.

According to the cell culture medium for culturing organoid of thepresent embodiment, tissue can be cultured over a long period of timethat does not substantially contain EGF or p38 inhibitor but containsepithelial stem cells, epithelial cells or epithelial tumor cellsderived from mammals, including humans, or tissue at least containingany one of these cells, or tissue unable to be cultured in the priorart.

In addition, according to the cell culture medium for culturing organoidof the present embodiment, an organoid can be formed with highefficiency from epithelial stem cells, epithelial cells or epithelialtumor cells derived from mammals, including humans, tissue at leastcontaining any one of these cells, or tissue unable to be cultured inthe prior art.

In addition, the differentiation ability of epithelial stem cellscultured using the cell culture medium for culturing organoid of thepresent embodiment can be maintained over a long period of time and thetumor incidence thereof is extremely low.

In the present description, epithelial cells include differentiatedepithelial cells and epithelial stem cells acquired from epithelialtissue. “Epithelial stem cells” refers to stem cells derived fromepithelial tissue that have the ability to self-replicate anddifferentiate into differentiated epithelial cells for a long period oftime. Examples of epithelial tissue include the cornea, oral mucosa,skin, conjunctiva, bladder, uriniferous tubules, kidneys, digestiveorgans (esophagus, stomach, duodenum, small intestine (including thejejunum and ileum) and large intestine (including the colon)), liver,pancreas, mammary glands, saliva glands, lacrimal glands, prostategland, hair roots, trachea and lungs. Among these, the cell culturemedium for culturing organoid of the present embodiment is particularlypreferably used to culture epithelial cells derived from digestiveorgans (esophagus, stomach, duodenum, small intestine (including thejejunum and ileum) and large intestine (including the colon)), liver andpancreas.

In addition, in the present description, “epithelial tumor cells” referto the aforementioned cells derived from epithelial tissue that havebecome tumorigenic.

In the present description, an “organoid” refers to a three-dimensionalcellular organ that is self-organized by accumulating cells at highdensity in a controlled space.

In the present description, “substantially not containing” refers to notcontaining a specific component at all or only containing a specificcomponent at a concentration at which the function thereof is notdemonstrated.

Accordingly, “substantially not containing EGF or p38 inhibitor” refersto not containing EGF or p38 inhibitor at all or only containing EGF andp38 inhibitor at a concentration at which inhibition of the expressionof EGFR by EGF and inhibition of differentiation and cell death causedby p38 inhibitor do not occur.

The cell culture medium for culturing organoid of the present embodimentat least contains one type of factor selected from the group consistingof IGF1, FGF2 and epiregulin, and whether any one of the aforementionedthree types of factors are contained can be suitably selectedcorresponding to the type of cells or tissue cultured. Among these, thecell culture medium for culturing organoid of the present embodimentpreferably contains IGF1 and epiregulin, FGF2 and epiregulin or IGF1 andFGF2 and more preferably contains IGF1 and FGF2. As a result ofcontaining the aforementioned factors, epithelial stem cells, epithelialcells or epithelial tumor cells derived from mammals, including humans,tissue at least containing these cells, or tissue unable to be culturedin the prior art, can be cultured for a long period of time withoutsubstantially containing EGF and p38 inhibitor. In addition, an organoidcan be formed with high efficiency from epithelial stem cells,epithelial cells or epithelial tumor derived from mammals, includinghumans, tissue at least containing any one of these cells, or tissueunable to be cultured in the prior art.

The following provides a detailed explanation of constituents of thecell culture medium for culturing organoid of the present embodiment.

<Cell Culture Basal Medium>

All types of serum-free cell culture basal media are included in thecell culture medium for culturing organoid of the present embodiment.The cell culture medium for culturing organoid of the present embodimentis preferably for animal cells or human cells.

Examples of this serum-free cell culture basal media include prescribedsynthetic media buffered with a carbonic acid-based buffering solutionto pH 7.2 to pH 7.6. More specifically, examples include glutamine,insulin, B27 supplement (Thermo Fisher), N-acetyl-L-cysteine (Wako PureChemical Industries), penicillin or streptomycin and Dulbecco's modifiedEagle medium/Ham's F-12 mixed medium supplemented with transferrin(Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12)).

In addition, other examples include Roswell Park Memorial Instituted(RPMI) 1640 medium, DMEM/F12 and Advanced RPMI medium instead ofadvanced Dulbecco's modified Eagle medium/Ham's F-12 mixed medium.

The aforementioned cell culture medium for culturing organoid of thepresent embodiment does not substantially contain indeterminatecomponents such as fetal bovine serum (FBS) or fetal calf serum.

In addition, the aforementioned cell culture medium for culturingorganoid may contain 5% serum.

<Wnt Agonist>

In the present description, a “Wnt agonist” refers to a chemical agentthat activates T-cell factor (TCF)/lymphoid enhancer factor(LEF)-mediated transcription in cells. Accordingly, Wnt agonists are notlimited to Wnt family proteins, but also include Wnt agonists thatactivated by bonding to members of the frizzled receptor family,intracellular β-catenin destruction inhibitors and TCF/LEF activatingsubstances. The Wnt agonist is preferably at least one type selectedfrom the group consisting of Wnt protein, R-spondin and GSK-3βinhibitor.

The Wnt agonist stimulates Wnt activity in cells by at least 10%,preferably at least 20%, more preferably at least 30%, even morepreferably at least 50% and particularly preferably at least 90% incomparison with the level of Wnt activity in the absence of the Wntagonist. Wnt activity can be investigated using a method known bypersons with ordinary skill in the art such as by measuring Wnttranscription activity with pTOPFLASH and pFOPFLASH Tcf luciferasereporter constructs (reference document: Korinek, et al., 1997, Science275: 1784-1787).

The Wnt agonist is preferably contained when culturing epithelial stemcells, epithelial cells, epithelial tumor cells or tissue at leastcontaining any one of these cells. The Wnt agonist contained in the cellculture medium for culturing organoid of the present embodiment morepreferably includes a complex of Wnt protein and afamin and even morepreferably contains both a complex of Wnt protein and afamin, andR-spondin. Epithelial stem cells or epithelial cells are able formorganoids with high efficiency as a result of the cell culture mediumfor culturing organoid of the present embodiment containing a complex ofWnt protein and afamin, and R-spondin.

[Wnt Protein]

There are no particular limitations on the origin of the Wnt proteinserving as a type of Wnt agonist and Wnt protein derived from variousorganisms can be used. Among these, Wnt protein derived from mammals ispreferable. Examples of mammals include humans, mice, rats, cows, pigsand rabbits. Examples of mammalian Wnt protein include Wnt1, Wnt2,Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a,Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11 and Wnt16. A plurality oftypes of Wnt protein may be used in combination in the cell culturemedium for culturing organoid of the present embodiment.

An example of a method for producing Wnt protein consists of producingWnt protein using Wnt protein-expressing cells. There are no particularlimitations on the origin of the Wnt protein-expressing cells (such asspecies or culture form) and are only required to be cells that expressWnt protein stably and may be cells that express Wnt proteintransiently. Examples of Wnt protein-expressing cells include L dellsstably expressing mouse Wnt3a (ATCC CRL-2647) and L cells stablyexpressing mouse Wnt5a (ATCC CRL-2814). In addition, Wntprotein-expressing cells can also be produced using a known generecombination technology. Namely, Wnt protein-expressing cells can beproduced by inserting DNA encoding a desired Wnt protein into a knownexpression vector and introducing the resulting expression vector intosuitable host cells. The base sequence of the gene that encodes thedesired Wnt protein can be acquired from a known database such asGenBank.

The Wnt protein expressed by Wnt protein-expressing cells may be afragment of Wnt protein and may contain an amino acid sequence otherthan the amino acid sequence of Wnt protein provided it has Wntactivity. There are no particular limitations of the amino acid sequenceother than amino acid sequence of Wnt protein, and an example thereof isthe amino acid sequence of an affinity tag. In addition, the amino acidsequence of the Wnt protein is not required to completely coincide withan amino acid sequence acquired from a known database such as GenBank,but rather may be an amino acid sequence that is substantially identicalto an amino acid sequence able to be acquired from a known databaseprovided it has Wnt activity.

Examples of amino acid sequences that are substantially identical to anamino acid sequence of Wnt protein able to be acquired from a knowndatabase such as GenBank include amino acid sequences in which one to aplurality of amino acids have been deleted, substituted or added.

An “amino acid sequence in which one to a plurality of amino acids havebeen deleted, substituted or added” refers to an amino acid sequence inwhich amino acids in roughly a number thereof that can be deleted,substituted or added (preferably 10 or less, more preferably 7 or lessand even more preferably 6 or less) have been deleted, substituted oradded by a known mutant protein production method such as site-specificmutagenesis.

In addition, examples of substantially identical amino acid sequencesinclude amino acid sequences in which identity with an amino acidsequence able to be acquired from a known database is at least 80% ormore, preferably at least 85% or more, more preferably at least 90% ormore, even more preferably at least 92% or more, particularly preferablyat least 95% or more, and most preferably at least 99% or more.

Wnt protein activity can be confirmed by, for example, TCF reporterassay. In general, a TCF reporter assay refers to a method consisting ofintroducing a luciferase gene having a binding sequence for T-cellfactor (TCF), which is a transcription factor that is specificallyactivated when a Wnt signal enters a cell and simply evaluating theintensity of Wnt protein activity based on the luminescence of theluciferase (reference document: Molenaar, et al., Cell, 86, 391, 1996).An example of a method other than the TCF reporter assay consists ofutilizing the stabilization of β-catenin in a cell when a Wnt signalenters that cell and then quantitatively evaluating the amount ofβ-catenin by western blotting (reference document: Shibamoto, et al.,Gene to cells, 3, 659, 1998). In addition, an example of a method thatcan be used to evaluate the activity of Wnt protein that imparts asignal to cells via a non-canonical pathway in the manner of Wnt5aconsists of evaluating the phosphorylation of an intracellular adapterprotein in the form of Dvl2 (reference document: Kikuchi, et al., EMBOJ., 29, 3470, 2010).

[R-Spondin]

Examples of a type of Wnt agonist in the form of R-spondin includemembers of the R-spondin family composed of R-spondin 1, R-spondin 2,R-spondin 3 and R-spondin 4. The R-spondin family consists of secretoryproteins that are known to be involved in activation and control of theWnt signal transduction pathway. A plurality of types of R-spondin maybe used in combination in the cell culture medium for culturing organoidof the present embodiment.

The R-spondin may be a fragment of R-spondin or contain an amino acidsequence other than the amino acid sequence of R-spondin provided it hasR-spondin activity.

[Content]

The concentration of Wnt protein contained in the cell culture mediumfor culturing organoid of the present embodiment is preferably 50 ng/mLor more, more preferably 10 ng/mL to 10 μg/mL, even more preferably 200ng/mL to 1 μg/mL and particularly preferably 300 ng/mL to 1 μg/mL. Wntagonist is preferably added to the culture medium every two days whenculturing epithelial stem cells, and the cell culture medium forculturing organoid is preferably replaced with fresh medium every fourdays.

[GSK-3β Inhibitor]

Known GSK-3β inhibitors include CHIR-99021, CHIR-98014 (Sigma-Aldrich)lithium (Sigma), kenpaullone (Biomol. International, Leost, M., et al.(2000), Eur. J. Biochem. 287, 5983-5994), 6-bromoindirubin-30-acetoxime(Meyer, L., et al. (2003) Chem. Biol. 10, 1255-1266), SB 218763 and SB415286 (Sigma-Aldrich) as well as members of the FRAT family andFRAT-derived peptides that inhibit interaction between GSK-3 and axin.An overview thereof is indicated in Meijer, et al. (2004) Trends inPharmacological Sciences 25, 471-480, which is incorporated in thepresent description by reference. Methods and assays for determining thelevel of GSK-3β inhibitor are known among persons with ordinary skill inthe art and examples thereof include the method and assay described inLiao, et al. (2004), Endocrinology, 145(6), 2941-2949.

<Afamin>

In the present description, “afamin” refers to a glycoprotein belongingto the albumin family that is known to be present in blood or bodyfluid. Afamin derived from animals from which the serum is sampled iscontained in serum normally added to medium used for cell culturing.Since serum contains impurities and the like other than afamin, afaminis preferably used along without using serum in the cell culture mediumfor culturing organoid of the present embodiment.

There are no particular limitations on the source of the afamincontained in the cell culture medium for culturing organoid of thepresent embodiment, and afamin derived from various organisms can beused. Among these, afamin derived from mammals is preferable. Examplesof mammals include the same as those in the previously described sectionentitled, “Wnt Protein”. Main amino acid sequences of mammalian afaminalong with the base sequences of genes encoding those amino acidsequences can be acquired in a known database such as GenBank. Forexample, the amino acid sequence of human afamin is registered inGenBank under the accession number AAA21612 and the base sequence of thegene encoding that sequence is registered in under the accession numberL32140, the amino acid sequence of bovine afamin is registered under theaccession number DAA28569, and the base sequence of the gene encodingthat sequence is registered under the accession number GF060968.

The afamin contained in the cell culture medium for culturing organoidof the present embodiment may be purified according to a known method inthe case of naturally-occurring afamin contained in serum and the likeor may be in the form of recombinant afamin. Recombinant afamin can beproduced by suitably using a known gene recombination technology. Anexample of a method for producing recombinant afamin consists ofinserting DNA encoding afamin into a known expression vector,introducing the resulting expression vector into a suitable host cell toexpress recombinant afamin, and then purifying the recombinant afaminusing a known purification method. The recombinant afamin may be afaminto which an affinity tag has been added. There are no particularlimitations on the added affinity tag and can be suitably selected foruse from among known affinity tags. The affinity tag is preferably anaffinity tag recognized by a specific antibody, and examples thereofinclude FLAG tag, MYC tag, HA tag and V5 tag.

The aforementioned Wnt protein has strong hydrophobicity as a result ofa specific serine residue being modified with a fatty acid (palmitoleicacid). Consequently, Wnt protein is widely known to be extremelydifficult to purify and store as a result of being susceptible toagglutination and degeneration when in an aqueous solution.

On the other hand, this modification of a specific serine residue byfatty acid is essential for the physiological activity of Wnt proteinand has been reported to be involved in bonding with members of thefrizzled receptor family.

In addition, in an aqueous solution, there are also findings indicatingthat Wnt protein bonds with afamin at a 1:1 ratio to form a complex andbecome soluble while maintaining a high level of physiological activity(Active and water-soluble form of lipidated Wnt protein is maintained bya serum glycoprotein in afamin/α-albumin, Mihara, E., Hirai, H.,Yamamoto, H., Tamura-Kawakami, K., Matano, M., Kikuchi, A., Sato, T.,Takagi, J., Elife, 2016 Feb. 23; 5).

On the basis of these findings, Wnt-protein-afamin complex may beproduced according to a method consisting of culturing cells expressingboth Wnt protein and afamin, or Wnt protein-afamin complex may beproduced according to a method consisting of co-culturing Wntprotein-expressing cells and afamin-expressing cells. Activity of Wntprotein in the Wnt protein-afamin complex can be evaluated using methodssimilar to those used for the aforementioned “Wnt protein”.

Although there are no particular limitations thereon, the concentrationof afamin in the cell culture medium for culturing organoid of thepresent embodiment is preferably 50 ng/mL to 10 μg/mL, more preferably100 ng/mL to 1 μg/mL, and even more preferably 300 μg/mL to 1 μg/mL

<Insulin-Like Growth Factor 1 (IGF1)>

In general, “insulin-like growth factor 1 (IGF1)” is also referred to assomatomedin C and is a factor that is secreted by stimulation of growthhormone (GH) primarily in the liver. Nearly all cells of the body (andparticularly muscle, bone, liver, kidney, nerve, skin and lung cells)are known to be affected by IGF1. In addition to demonstrating aninsulin-like effect, IGF1 has functions that regulate cell growth (andparticularly, nerve cells) and development as well as cell DNAsynthesis.

Although there are no particular limitations thereon, the concentrationof IGF1 contained in the cell culture medium for culturing organoid ofthe present embodiment is preferably 5 ng/mL to 1 μg/mL, more preferably10 ng/mL to 1 μg/mL, and even more preferably 50 ng/mL to 500 ng/mL. Asa result of making the concentration of IGF1 contained in the cellculture medium for culturing organoid of the present embodiment to bewithin the aforementioned ranges, epithelial stem cells, epithelialcells or epithelial tumor cells derived from mammals, including humans,tissue at least containing any one of these cells, or tissue unable tobe cultured in the prior art, can be cultured for a long period of timewithout substantially containing EGF and p38 inhibitor.

In addition, an organoid can be formed with high efficiency fromepithelial stem cells, epithelial cells or epithelial tumor cellsderived from mammals, including humans, tissue at least containing anyone of these cells, or tissue unable to be cultured in the prior art.

In addition, during culturing of stem cells, IFG1 is preferably added tothe culture medium every two days, and the culture medium is preferablyreplaced with fresh medium every four days.

<Fibroblast Growth Factor 2 (FGF2)>

In general, fibroblast growth factor 2 (FGF2) refers to a basicfibroblast growth factor that has the functions of promoting the growthof vascular endothelial cells and organizing into a tubular structure,or in other words, promoting vascularization by bonding with fibroblastgrowth factor receptor (FGFR). In addition, human FGF2 is known to havetwo isoforms consisting of low molecular weight form (LWL) and highmolecular weight form (HWL). LWL is mainly present in cytoplasm and actsin the manner of an autocrine, while on the other hand, HWL is presentin the cell nucleus and demonstrates activity by an intracrine mechanismthat acts within cells.

Although there are no particular limitations thereon, the concentrationof FGF2 contained in the cell culture medium for culturing organoid ofthe present embodiment is preferably 5 ng/mL to 1 μg/mL, more preferably10 ng/mL to 1 μg/mL, and even more preferably 50 ng/mL to 500 ng/mL. Asa result of making the concentration of FGF2 contained in the cellculture medium for culturing organoid of the present embodiment to bewithin the aforementioned ranges, epithelial stem cells, epithelialcells or epithelial tumor cells derived from mammals, including humans,tissue at least containing any one of these cells, or tissue unable tobe cultured in the prior art, can be cultured for a long period of timewithout substantially containing EGF and p38 inhibitor.

In addition, an organoid can be formed with high efficiency fromepithelial stem cells, epithelial cells or epithelial tumor cellsderived from mammals, including humans, tissue at least containing anyone of these cells, or tissue unable to be cultured in the prior art.

In addition, during culturing of stem cells, FGF2 is preferably added tothe culture medium every two days, and the culture medium is preferablyreplaced with fresh medium every four days.

<Epiregulin (EREG)>

In general, epiregulin (EREG) refers to an EGF-like growth factor thatspecifically binds with ErbB1 and ErbB4 among receptors (ErbB1 to ErbB4)of the tyrosine kinase (ErbB) family. EREG is known to stimulate growthof keratin-producing cells, liver cells, fibroblasts and vascularendothelial cells. In addition, EREG is mainly expressed in carcinomasof the bladder, lungs, kidneys and colon, placenta and peripheral whiteblood cells.

Although there are no particular limitations thereon, the concentrationof EREG contained in the cell culture medium for culturing organoid ofthe present embodiment is preferably 5 ng/mL to 1 μg/mL, more preferably10 ng/mL to 1 μg/mL, and even more preferably 50 ng/mL to 500 ng/mL. Asa result of making the concentration of EREG contained in the cellculture medium for culturing organoid of the present embodiment to bewithin the aforementioned ranges, epithelial stem cells, epithelialcells or epithelial tumor cells derived from mammals, including humans,tissue at least containing any one of these cells, or tissue unable tobe cultured in the prior art, can be cultured for a long period of timewithout substantially containing EGF and p38 inhibitor.

In addition, an organoid can be formed with high efficiency fromepithelial stem cells, epithelial cells or epithelial tumor cellsderived from mammals, including humans, tissue at least containing anyone of these cells, or tissue unable to be cultured in the prior art.

In addition, during culturing of stem cells, EREG is preferably added tothe culture medium every two days, and the culture medium is preferablyreplaced with fresh medium every four days.

<BMP Inhibitor>

Bone morphogenetic protein (BMP) binds as a dimer ligand to a receptorcomplex consisting of two different types of receptors ofserine/threonine kinase, type I and type II receptors. Type II receptorphosphorylates the type I receptor thereby resulting in activation ofthe receptor kinase. This type I receptor subsequently phosphorylates aspecific receptor substrate (SMAD) resulting in induction oftranscription activity by a signal transduction pathway. In general, BMPinhibitor prevents or inhibits the formation of complex that neutralizesBMP activity, for example, and is a chemical agent that binds to BMPmolecules in order to form a receptor that neutralizes BMP activity. Inaddition, BMP inhibitor also binds to BMP receptor, for example, toprevent or inhibit binding of BMP molecules to receptors, and is achemical agent that acts as an antagonist or inverse agonist.

BMP inhibitor has inhibitory activity that is preferably 50% or more,more preferably 70% or more, even more preferably 80% or more andparticularly preferably 90% or more than the level of BMP activity inthe absence of this inhibitor. BMP inhibitory activity can be evaluatedby measuring BMP transcription activity using a method known amongpersons with ordinary skill in the art (reference document: Zilberberg,et al., BMC Cell Biol., 41, 2007).

A naturally-occurring BMP binding protein is preferable for the BMPinhibitor contained in the cell culture medium for culturing organoid ofthe present embodiment, and examples thereof include chordin-likeproteins such as noggin, gremlin, chordin or chordin domain,follistatin-related proteins such as follistatin or follistatin domain,DAN-like proteins such as DAN or DAN cysteine knot domain,sclerostin/SOST, decorin and α-2 macroglobulin.

Chordin-like protein or DAN-like protein is preferable for the BMPinhibitor contained in the cell culture medium for culturing organoid ofthe present embodiment and chordin-like protein is more preferable.Noggin is preferable for the chordin-like protein. Chordin-like proteinand DAN-like protein are diffusing proteins that are able to inhibit BMPmolecules from approaching signal transduction receptors by binding toBMP molecules with various degrees of affinity. In the case of culturingepithelial stem cells, these BMP inhibitors are able to prevent loss ofstem cells by adding to cell culture medium for culturing organoid.

The concentration of BMP inhibitor contained in the cell culture mediumfor culturing organoid of the present embodiment is preferably 10 ng/mLto 100 ng/mL, more preferably 20 ng/mL to 100 ng/mL, and even morepreferably 500 ng/mL to 100 ng/mL. During culturing of stem cells, BMPinhibitor is preferably added to the culture medium every two days, andthe culture medium is preferably replaced with fresh medium every fourdays.

<TGF-β Inhibitor>

Transforming growth factor β (TGF-β) is a type of growth factor that isproduced in nearly all cells such as those of the kidneys, bone marrowand platelets. There are five subtypes of TGF-β (β1-β5). In addition,TGF-β is known to promote the growth of fibroblasts along with thesynthesis and growth of connective tissue in the manner of collagenwhile also acting suppressively on epithelial cell growth andosteoclasts. In general, TGF-β inhibitor is a chemical agent thatprevents or inhibits binding of TGF-β to TGF-β receptors and binds withTGF-β to form a complex that neutralizes TGF-β activity. In addition,TGF-β inhibitor is a chemical agent that prevents or inhibits binding ofTGF-β to TGF-β receptors by binding with TGF-β receptors, for example,thereby acting as an agonist or antagonist.

TGF-β inhibitor has inhibitory activity that is preferably 50% or more,more preferably 70% or more, even more preferably 80% or more andparticularly preferably 90% or more than the level of TGF-β activity inthe absence of this inhibitor. TGF-β activity can be evaluated by amethod known among persons with ordinary skill in the art. An example ofan evaluation system consists of a cell assay of stably transfectedcells using a reporter construct containing human PAI-1 promoter or Smadbinding site acting on luciferase reporter gene (reference document: DeGouville, et al., Br. J. Pharmacol., 145(2): 166-177, 2005).

Examples of TGF-β inhibitor contained in the cell culture medium forculturing organoid of the present embodiment include A83-01(3-(6-methylpyridin-2-yl)-1-phenylthiocarbamoyl-4-quinolin-ylpyrazole),ALK5 inhibitor I (3-(pyridin-2-yl)-4-(4-quinonyl)-1H-pyrazole),LDN1931189(4-(6-(4-piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline),SB431542(4-[4-(1,3-benzodioxol-5-yl)-5-pyridin-2-yl-1H-imidazol-2-yl]benzamide),SB-505124(2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridinehydrochloride hydrate), SD-208(2-(5-chloro-2-fluorophenyl)pteridin-4-yl)pyridin-4-yl-amine), SB-525334(6-[2-(1,1-dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-4-yl]quinoxaline)LY-364947 (4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline), LY2157299(4-[2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-quinoline-6-carboxylic acid amide), TGF-β RI kinase inhibitor II 616452(2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine), TGF-βRI kinase inhibitor III 616453(2-(5-benzo[1,3]dioxol-4-yl-2-tert-butyl-1H-imidazol-4-yl)-6-methylpyridineHCl), TGF-β RI kinase inhibitor IX 616463(4-((4-((2,6-dimethylpyridin-3-yl)oxy)pyridin-2-yl)amino)benzenesulfonamide), TGF-β RI kinase inhibitor VII 616458(1-(2-((6,7-dimethoxy-4-quinolyl)oxy)-(4,5-dimethylphenyl)-1-ethanone),TGF-β RI kinase inhibitor VIII 616459(6-(2-tert-butyl-5-(6-methyl-pyridin-2-yl)-1H-imidazol-4-yl)-quinoxaline),AP12009 (TGF-β2 antisense compound “Trabedersen”), Belagenpumatucel-L(TGF-β2 antisense gene-modified allogenic tumor cell vaccine), CAT-152(Glaucoma-lerdelimumab (anti-TGF-β2 monoclonal antibody), CAT-192(Metelimumab (human IgG4 monoclonal antibody that neutralizes TGF-β1),and GC-1008 (anti-TGF-β monoclonal antibody). A83-01 is preferable amongthe TGF-β inhibitors contained in the cell culture medium for culturingorganoid of the present embodiment.

The concentration of TGF-β contained in the cell culture medium forculturing organoid of the present embodiment is preferably 10 nM to 10μM, more preferably 500 nM to 5 μM and even more preferably 500 nM to 2μM. During culturing of stem cells, TGF-β inhibitor is preferably addedto the culture medium every two days, and the culture medium ispreferably replaced with fresh medium every four days.

<Other Components>

The cell culture medium for culturing organoid of the present embodimentmay also contain Rho kinase (Rock) inhibitor. Examples thereof includeY-27632 ((R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride hydrate), fasudil (HA1077)(5-(1,4-diazepan-1-ylsulfonyl)isoquinoline), and H-1152((S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolyl)sulfonyl]-hexahydro-1H-1,4-diazepinedihydrochloride). In the case of using Y-27632 as a Rock inhibitor,Y-27632 is preferably added for the first two days of culturing stemcells dispersed in single cells. The concentration of Y-27632 containedin the cell culture medium for culturing organoid of the presentembodiment is preferably 10 μM.

Gastrin (or a suitable substitute such as Leu15-gastrin) is furtheradded to the cell culture medium for culturing organoid of the presentembodiment. The concentration of gastrin (or suitable substitutethereof) contained in the cell culture medium for culturing organoid ofthe present embodiment is may be, for example, 1 ng/mL to 10 μg/mL, 1ng/mL to 1 μg/mL or 5 ng/mL to 100 ng/mL.

The cell culture medium for culturing organoid of the present embodimentmay also further contain at least one type of amino acid. Examples ofamino acids contained in the cell culture medium for culturing organoidof the present embodiment include L-alanine, L-arginine, L-asparagine,L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-glutamine,L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine,L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan,L-tyrosine, L-valine and combinations thereof. In general, theconcentration of L-glutamine contained in the cell culture medium is0.05 g/L to 1 g/L (and normally, 0.1 g/L to 0.75 g/L). In addition, theconcentration of other amino acids contained in the cell culture mediumis 0.001 g/L to 1 g/L (and normally, 0.01 g/L to 0.15 g/L). The aminoacids may be of a synthetic origin.

The cell culture medium for culturing organoid of the present embodimentmay further contain at least one type of vitamin. Examples of vitaminscontained in the cell culture medium for culturing organoid of thepresent embodiment include thiamine (vitamin B1), riboflavin (vitaminB2), niacin (vitamin B3), calcium D-pantothenate (vitamin B5),pyridoxal/pyridoxamine/pyridoxine (vitamin B6), folic acid (vitamin B9),cyanocobalamin (vitamin B12), ascorbic acid (vitamin C), calciferol(vitamin D2), DL-α-tocopherol (vitamin E), biotin (vitamin H) andmenadione (vitamin K).

The cell culture medium for culturing organoid of the present embodimentmay further also contain at least one type of inorganic salt. Theinorganic salt contained in the cell culture medium for culturingorganoid of the present embodiment is for assisting in maintaining theosmotic equilibrium of cells as well as assisting in regulating themembrane potential thereof. Specific examples of inorganic salts includesalts of calcium, copper, lead, magnesium, potassium, sodium and zinc.Salts are normally be used in the form of chlorides, phosphates,sulfates, nitrates and carbonates. Moreover, specific examples thereofinclude CaCl₂, CuSO₄-5H₂O, Fe(NO₂)-9H₂O, FeSO₄-7H₂O, MgCl, MgSO₄, KCl,NaHCO₃, NaCl, Na₂HPO₄, Na₂HPO₄—H₂O and ZnSO₄-7H₂O.

The cell culture medium for culturing organoid of the present embodimentmay further also contain at least one type of sugar capable of servingas a carbon energy source. Examples of sugars contained in the cellculture medium for culturing organoid of the present embodiment includeglucose, galactose, maltose and fructose. Among these, glucose ispreferable and D-glucose (dextrose) is particularly preferable for thesugar. The concentration of sugar contained in the cell culture mediumfor culturing organoid of the present embodiment is preferably 1 g/L to10 g/L.

The cell culture medium for culturing organoid of the present embodimentmay also further contain at least one type of trace element. Examples oftrace elements contained in the cell culture medium for culturingorganoid of the present embodiment include barium, bromium, cobalt,iodine, manganese, chromium, copper, nickel, selenium, vanadium,titanium, germanium, molybdenum, silicon, iron, fluorine, silver,rubidium, tin, zirconium, cadmium, zinc, aluminum and ions thereof.

The cell culture medium for culturing organoid of the present embodimentmay also further contain at least one type of supplementary chemicalagent. Examples of such supplementary chemical agents include nutrientsor growth factors that have been reported to improve stem cellculturing, such as cholesterol, transferrin, albumin, insulin,progesterone, putrescine, selenite or other factors.

<<Method for Culturing Epithelial Stem Cells, Epithelial Cells,Epithelial Tumor Cells or Tissue Containing these Cells>>

First Embodiment

The culture method according to the first embodiment of the presentinvention is a method for culturing epithelial stem cells, epithelialcells, epithelial tumor cells or tissue at least containing any one ofthese cells, provided with: an extracellular matrix preparation step forpreparing an extracellular matrix, an adhesion step for adheringepithelial stem cells, epithelial cells, epithelial tumor cells ortissue at least containing any one of these cells onto the extracellularmatrix, and an organoid formation step for forming an organoid by addingthe aforementioned cell culture medium for culturing organoid andculturing the epithelial stem cells, the epithelial cells, theepithelial tumor cells or tissue at least containing any one of thesecells, after the cell adhesion step.

According to the culture method of the present embodiment, epithelialstem cells, epithelial cells or epithelial tumor cells derived frommammals, including humans, tissue at least containing any one of thesecells, or tissue unable to be cultured in the prior art, can be culturedfor a long period of time. In addition, an organoid can be formed withhigh efficiency from epithelial stem cells, epithelial cells orepithelial tumor cells derived from mammals, including humans, tissue atleast containing any one of these cells, or tissue unable to be culturedin the prior art.

The following provides a detailed explanation of each step in theculture method of the present embodiment.

[Extracellular Matrix Preparation Step]

In general, an “extracellular matrix (ECM)” refers to a supramolecularstructure present outside the cells of a living organism. This ECMserves as a scaffold for the growth of epithelial stem cells, epithelialtumor cells or tissue containing these cells.

ECM contains various polysaccharides, water, elastin and glycoproteins.Examples of glycoproteins include collagen, entactin (nidogen),fibronectin and laminin.

An example of a method used to prepare ECM is a method that usesconnective tissue cells. More specifically, after having culturedECM-producing cells such as fibroblasts, these cells are extractedfollowed by the addition of epithelial stem cells, epithelial cells,epithelial tumor cells or tissue containing these cells to enables theuse thereof as scaffold.

Examples of ECM-producing cells include osteoclasts mainly producingcollagen and proteoglycan, fibroblasts mainly producing type IVcollagen, laminin, interstitial proteoglycan and fibronectin, and colonmyofibroblasts mainly producing collagen (type I, type III and type IV),chondroitin sulfate proteoglycans, hyaluronic acid, fibronectin andtenascin C. Alternatively, commercially available ECM may also be used.Examples of commercially available ECM include Extracellular MatrixProtein (Invitrogen), and basement membrane preparations derived fromEngelbreth-Holm-Swarm (EHS) mouse sarcoma cells (such as Matrigel®manufactured by BD Biosciences). Synthetic ECM such as ProNectin (SigmaZ378666) may also be used. In addition, mixtures of naturally-occurringECM and synthetic ECM may be used.

In the case of using ECM to culture stem cells, the long-term survivalof stem cells and continued survival of undifferentiated stem cells canbe enhanced. In the absence of ECM, stem cell cultures are unable to becultured over a long period of time and continued survival ofundifferentiated stem cells is not observed. Moreover, when ECM ispresent, organoids can be cultured that are unable to be cultured in theabsence of ECM.

ECM usually sinks to the bottom of a dish in which cells are suspended.For example, when ECM coagulates at 37° C., the aforementioned cellculture medium for culturing organoid may be added and used by diffusingin the ECM. Cells present in the medium adhere to the ECM by interactingwith integrin, for example, as a result of interacting with the surfacestructure of the ECM.

ECM may be coated onto a culture vessel and the like. In the case ofusing fibronectin, the ratio coated onto the culture vessel as ECM ispreferably 1 μg/cm² to 250 μg/cm², more preferably 1 μg/cm² to 150μg/cm², and even more preferably 8 μg/cm² to 125 μg/cm².

[Adhesion Step]

Continuing, epithelial stem cells, epithelial cells, epithelial tumorcells or tissue at least containing any one of these cells are prepared.Examples of epithelial stem cells, epithelial cells, epithelial tumorcells and tissue at least containing any one of these cells used in theculture method of the present embodiment include the same cells andtissue as those in the previously described section entitled, <<CellCulture Medium for Culturing Organoid>>.

Examples of methods used to prepare epithelial cells from theaforementioned tissue include methods known among persons with ordinaryskill in the art. For example, crypts can be isolated by allowing achelating agent and isolated tissue to stand at a constant temperature.This tissue is then washed followed by detaching the epithelial celllayer from the submucosal layer on a glass slide and slicing into thinsections. Subsequently, the thin sections are allowed to stand at aconstant temperature in trypsin or preferably a solution containing atleast one of EDTA and EGTA followed by separating the undigested tissuefragments and single cells derived from the crypts using, for example,at least one of filtration and centrifugation. Instead of using trypsin,at least one type of other proteolytic enzyme such as collagen ordispase I may be used. A similar method is used to isolate pancreas orstomach fragments.

Examples of methods for isolating stem cells from epithelial tissueinclude methods known among persons with ordinary skill in the art. Stemcells express at least one of Lgr5 and Lgr6 on the surface thereof (Lgr5and Lgr6 belong to a superfamily of large G protein coupled receptors(GPCR)). An example of an isolation method consists of preparing a cellsuspension from the epithelial tissue, contacting this cell suspensionwith a chemical substance that binds with at least one of Lgr5 and Lgr6,separating the chemical substance that binds with at least one of Lgr5and Lgr6, and isolating stem cells from this binding compound.

Specific examples of chemical substances that bind with at least one ofLgr5 and Lgr6 include antibodies, and more specifically, monoclonalantibodies that specifically recognize at least one of Lgr5 and Lgr6 andbind thereto (such as monoclonal antibodies containing mouse and ratmonoclonal antibodies). Use of such antibodies makes it possible toisolate stem cells that express at least one of Lgr5 and Lgr6 using, forexample, magnetic beads or by passing through a fluorescence-activatedcell sorter.

Epithelial stem cells, epithelial cells, epithelial tumor cells ortissue at least containing any one of these cells that have beenisolated according to the aforementioned method are seeded and allowedto stand undisturbed on the cell matrix obtained in the aforementionedpreparation step. The seeded cells are able to adhere to ECM byinteracting with integrin, for example, as a result of interacting withthe surface structure of the ECM.

[Organoid Formation Step]

Continuing, after seeding the cells, the aforementioned cell culturemedium for culturing organoid is added before the cells dry followed byculturing the cells. The culture temperature is preferably 30° C. to 40°C. and more preferably at about 37° C. Culture time can be suitablyadjusted according to the cells used. An organoid can be formed afterroughly 1 to 2 weeks after the start of culturing. In addition, cellsthat were only able to be maintained and cultured for two to threemonths in the prior art can be maintained and cultured for a long periodof time of 3 months or longer (and preferably about 10 months) in thecase of the culture method of the present embodiment. Use of the culturemethod of the present embodiment makes it possible to maintaindifferentiation ability and suppress the frequency of tumorigenesis to alow level in the case of culturing the aforementioned stem cells.

In addition, culturing may be carried out under hypoxic condition duringthe organoid formation step. Culturing under hypoxic condition enablesorganoids to be formed with high efficiency from epithelial stem cells,epithelial cells or epithelial tumor cells derived from mammals,including humans, tissue at least containing any one of these cells, ortissue unable to be cultured in the prior art.

Hypoxic condition in the present embodiment preferably refer to anoxygen concentration of 0.1% to 15%, more preferably 0.3% to 10%, andeven more preferably 0.5% to 5%.

Second Embodiment

The culture method according to a second embodiment of the presentinvention is a method for culturing epithelial stem cells, epithelialcells, epithelial tumor cells or tissue at least containing any one ofthese cells; provided with: an extracellular matrix preparation step forpreparing an extracellular matrix, an adhesion step for adheringepithelial stem cells, epithelial cells, epithelial tumor cells ortissue at least containing any one of these cells onto the extracellularmatrix, and an organoid formation step for forming an organoid by addingthe cell culture medium for culturing organoid and culturing theepithelial stem cells, the epithelial cells, the epithelial tumor cellsor tissue at least containing any one of these cells under hypoxiccondition, after the cell adhesion step; wherein, the cell culturemedium for culturing organoid contains at least one type of componentselected from the group consisting of Wnt agonist composed of Wntprotein and R-spondin, mitogenic growth factor, bone morphogeneticprotein (BMP) inhibitor, transforming growth factor β (TGF-β) inhibitorand p38 inhibitor, and the oxygen concentration is preferably 0.1% to15%, more preferably 0.3% to 10%, and even more preferably 0.5% to 5%.

According to the culture method of the present embodiment, epithelialstem cells, epithelial cells or epithelial tumor cells derived frommammals, including humans, tissue at least containing any one of thesecells, or tissue unable to be cultured in the prior art, can be culturedfor a long period of time. In addition, an organoid can be formed withhigh efficiency from epithelial stem cells, epithelial cells orepithelial tumor cells derived from mammals, including humans, tissue atleast containing any one of these cells, or tissue unable to be culturedin the prior art.

Each step of the culture method of the present embodiment is the same asthat of the aforementioned first embodiment.

In addition, the cell culture medium for culturing organoid used in theculture method of the present embodiment contains at least one type ofcomponent selected from the group consisting of Wnt agonist composed ofWnt protein and R-spondin, mitogenic growth factor, BMP inhibitor, TGF-βinhibitor and p38 inhibitor. In addition, in the culture method of thepresent embodiment, suitable conditions can be selected from among aplurality of conditions corresponding to the cultured cells or type oftissue by preparing and culturing several types of cell culture mediafor culturing organoid having different compositions under hypoxic andnormoxic conditions (oxygen concentration of about 20%), therebyenabling organoids to be formed with high efficiency.

Examples of Wnt agonist, BMP inhibitor and TGF-β inhibitor include thesame as those exemplified in the previously described section entitled,<<Cell Culture Medium for Culturing Organoid>>. In addition, the Wntagonist used preferably forms a complex with afamin. In the culturemethod of the present embodiment, the use of a cell culture medium forculturing organoid containing a Wnt agonist composed of a complex of Wntprotein and afamin, and R-spondin makes it possible for form an organoidwith high efficiency from epithelial stem cells or epithelial tumorcells derived from mammals, including humans, tissue at least containingany one of these cells, or tissue unable to be cultured in the priorart.

In addition, the following provides an explanation of other constituentscontained in the cell culture medium for culturing organoid used in theculture method of the present embodiment.

(Mitogenic Growth Factor)

Examples of mitogenic growth factors contained in the cell culturemedium for culturing organoid used in the culture method of the presentembodiment include a family of growth factors such as epidermal growthfactor (EGF), transforming growth factor-α (TGF-α), brain-derivedneurotrophic factor (BDNF) or keratinocyte growth factor (KGF). Aplurality of types of these mitogenic growth factors may also be used incombination.

EGF is a potent mitogenic factor for various cultured ectodermal cellsand mesodermal cells that has a remarkable effect on differentiation ofsome fibroblasts into specific cells. EGF precursor is present as amembrane-bound molecule that generates a 53-amino acid peptide thatstimulates cells after being cleaved by proteolysis. Among these, EGF ispreferable for the mitogenic growth factor contained in theaforementioned cell culture medium for culturing organoid. Theconcentration of EGF contained in the cell culture medium for culturingorganoid of the present embodiment is preferably 5 ng/mL to 500 ng/mL,more preferably 100 g/mL to 400 ng/mL, and even more preferably 50 ng/mLto 200 ng/mL.

In addition, the content of EGF is the same as that of KGF in the caseof containing KGF in the aforementioned cell culture medium forculturing organoid. In the case of using a plurality of KGF such as KGF1and KGF2 (also known as FGF7 and FGF10), the total content of KGF ispreferably within the aforementioned ranges. When culturing stem cells,mitogenic growth factor is preferably added to the culture medium everytwo days and the culture medium is replaced with fresh medium every fourdays.

(p38 Inhibitor)

In the present description, “p38 inhibitor” refers to an arbitraryinhibitor that directly or indirectly down-regulates p38 signaltransduction. In general, p38 inhibitor binds to p38, for example andreduces the activity thereof p38 protein kinase constitutes a portion ofthe family of mitogen-activated protein kinases (MAPK). MAPK areserine/threonine-specific protein kinases that regulate various cellactivities such as gene expression, mitosis, differentiation, growth orcell death/apoptosis by responding to an extracellular stimulus such asstress or inflammatory cytokines. p38 MAPK exists as isoforms α, β, β2,γ and δ. In addition, p38 inhibitor is a chemical agent that binds to atleast one p38 isoform, for example, and reduces the activity thereof.

p38 inhibitor has inhibitory activity that is preferably 50% or more,more preferably 70% or more, even more preferably 80% or more andparticularly preferably 90% or more in comparison with the level of p38activity in the absence of this inhibitor. The inhibitory effects of p38inhibitor can be evaluated using a method known among persons withordinary skill in the art. Examples of such evaluation systems include amethod for detecting a phosphorylation site-specific antibody ofThr180/Tyr182 phosphorylation, biochemical recombinant kinase assay,tumor necrosis factor α (TNF-α) secretion assay, and Discover Rxhigh-throughput screening platform for p38 inhibition (such as thatmanufactured by Millipore or Sigma-Aldrich).

Examples of p38 inhibitors contained in the cell culture medium forculturing organoid of the present embodiment include SB-202190(4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole),SB-203580(4-[4-(4-fluorophenyl)-2-[4-(methylsulfinyl)phenyl]-1H-imidazol-5-yl]pyridine,VX-702(6-(N-carbamoyl-2,6-difluoroanilino)-2-(2,4-difluorophenyl)pyridine-3-carboxyamide),VX-745(5-=(2,6-dichlorophenyl)-2-[(2,4-difluorophenyl)thio]-6H-pyrimido[1,6-b]pyridazin-6-one),PD-169316(4-(4-fluorophenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)-1H-imidazole),RO-4402257(6-(2,4-difluorophenoxy)-2-{[3-hydroxy-1-(2-hydroxyethyl)propyl]amino}-8-methylpyrido(2,3-D)pyrimidin-7(8h)-one),and BIRB-796(1-[5-tert-butyl-2-(4-methylphenyl)pyrazol-3-yl]-3-[4-(2-morpholin-4-ylethoxy)naphthalen-1-yl]urea).

The concentration of p38 inhibitor contained in the aforementioned cellculture medium for culturing organoid is preferably 50 nM to 100 μM,more preferably 100 nM to 50 μM and even more preferably 100 nM to 10μM. When culturing stem cells, p38 inhibitor is preferably added to theculture medium every two days and the culture medium is replaced withfresh medium every four days.

<<Organoid>>

The organoid according a first embodiment of the present invention isobtained by the previously described culture method.

The organoid of the present embodiment can be used in regenerativemedicine, basic medical research on epithelial cells, drug responsescreening and drug development research using patient-derived epithelialorganoids.

<Applications>

In one embodiment thereof, the present invention provides the use of theaforementioned organoid for drug response screening, toxicity assays orregenerative medicine.

In the case of using the aforementioned organoid in drug responsescreening, the organoid is cultured in, for example, a multi-well platesuch as a 96-well plate or 384-well plate. Molecules are then identifiedthat have an effect on the organoid using a molecule library. Examplesof molecule libraries include an antibody fragment library, peptidephage display library, peptide library (such as LOPAP® available fromSigma-Aldrich, lipid library (available from BioMol), synthetic compoundlibrary (such as LOPAP® available from Sigma-Aldrich) or naturalcompound library (Specs available from TimTec). Moreover, a gene librarymay also be used. Examples of gene libraries include a cDNA library,antisense library, and siRNA or other non-coding RNA library. An exampleof a specific method consists of exposing cells to multipleconcentrations of a test chemical agent over a certain period of timefollowed by evaluating the culture at completion of exposure. Inaddition, although the organoid of the present embodiment specificallytargets epithelial tumor cells, it can also be used to identify chemicalagents not targeting organoids composed of normal cells.

Moreover, the organoid of the present embodiment can also be usedinstead of cell lines such as Caco-2 cells in toxicity assays carriedout on new candidate drugs or known or new dietary supplements.

Moreover, the organoid of the present embodiment can be used to culturepathogens such as norovirus for which there are currently no suitabletissue cultures or animal models.

In addition, the organoid of the present embodiment is useful in thefield of regenerative medicine in the repair of intestinal epitheliumfollowing radiation exposure or surgery, in the repair of intestinalepithelium of patients suffering from inflammatory bowel diseases suchas Crohn's disease or ulcerative colitis, or in the repair of intestinalepithelium of patients suffering from short bowel syndrome. Moreover,the organoid of the present embodiment is useful in the repair ofintestinal epithelium in patients with genetic diseases of the smallintestine or colon. In addition, the organoid of the present embodimentis useful in the field of regenerative medicine, for example, as a graftor portion thereof following pancreatectomy or for the treatment ofdiabetes such as type I diabetes or type II diabetes.

Although the following provides an explanation of the present inventionthrough examples thereof, the present invention is not limited to thefollowing examples.

EXAMPLES Example 1

(1) Preparation of Cell Culture Medium for Organoid Culturing

First, human recombinant R-spondin 1 (R&D Systems) was added to acommercially available Advanced DMEM/F-12 medium (Thermo FisherScientific) to a final concentration of 1 μg/mL followed by the additionof noggin (Peprotech) to a final concentration 100 ng/mL and A83-01(Tocris) to a final concentration of 500 nM. Moreover, a medium wasprepared in which culture supernatant derived from W-Wnt3a/HEK culturedin serum-containing medium was added to a final concentration of Wnt3aof 300 ng/mL (to be referred to as “WNRA medium”).

Moreover, at least one type among Epiregulin (Biolegend) to a finalconcentration of 500 ng/mL, IGF1 (Biolegend) to a final concentration of500 ng/mL, or FGF2 (Peprotech) to a final concentration of 50 ng/mL, wasadded to prepare media in which constituents were combined in the mannerindicated below.

-   -   WNRA+epiregulin medium    -   WNRA+IGF1 medium    -   WNRA+epiregulin+IGF1 medium    -   WNRA+epiregulin+FGF2 medium    -   WNRA+IGF1+FGF2 medium

In addition, medium serving as a control was also prepared in which EGF(Thermo Fisher Scientific) was added to a final concentration of 50ng/mL (to be referred to as “WNRA+EGF medium”). In addition, mediumserving as a reference example was prepared in which SB202190(Sigma-Aldrich) was further added to a final concentration of 10 μM (tobe referred to as “WNRAS+EGF medium”).

(2) Culturing of Epithelial Tumor Cells Derived from Colon Tumor

Lesion tissue from a colon tumor, or a portion at least 5 cm away fromthe colon tumor treated as normal mucosa, was sampled from colon tumorpatients provided with an explanation and from whom consent had beenobtained based on an ethics research protocol approved by the ethicscommittee of Keio University School of Medicine. Next, colon tumortissue containing epithelial tumor cells derived from human tumor tissuewas divided into epithelial tumor cells and residual tissue usingLiberase TH. The remaining tissue was further loosely dispersed withtrypsin. Next, the epithelial tumor cells were seeded in a 48-well platetogether with 25 μL of Matrigel® (BD Bioscience). The WNRA+EGF medium(medium used in conventional methods), WNRA+epiregulin medium, WNRA+IGF1medium, WNRA+epiregulin+IGF medium, WNRA+epiregulin+FGF2 medium orWNRA+IGF1+FGF2 medium prepared in (1) above were added in aliquots of250 μL each to the wells seeded with epithelial tumor cells followed byculturing at 37° C. and oxygen concentration of 20%. The medium wasreplaced every two days from the start of culturing. FIG. 1 shows imagesdepicting the states of the cultures on day 7 from the start of primaryculturing (passage 0). FIG. 2 is a graph quantifying the area in thewells occupied by intestinal stem cells for each medium.

Based on FIGS. 1 and 2 , organoid area was confirmed to increase andorganoids were confirmed to be able to be cultured with high efficiencyin comparison with WNRA+EGF medium regardless of the medium used. Inparticular, organoids were clearly determined to be able to be culturedwith high efficiency in the case of using “WNRA+IGF1+FGF2 medium”containing IGF1 and FGF2.

Example 2

(1) Preparation of Cell Culture Medium for Organoid Culturing

First, medium was prepared in which EGF (Thermo Fisher Scientific) to afinal concentration of 50 ng/mL, noggin (Peprotech) to a finalconcentration of 100 ng/mL and A83-01 (Tocris) to a final concentrationof 500 nM were added to a commercially available Advanced DMEM/F-12medium (Thermo Fisher Scientific) (and the resulting medium is alsoreferred to as “ENA medium”).

(2) Preparation of Epithelial Tumor Tissue Derived from Colon Tumor

Epithelial tumor cells were obtained using the methods as thosedescribed in sections (1) and (2) of Example 1. Next, the epithelialtumor cells were seeded in a 48-well plate together with 25 μL ofMatrigel® (BD Bioscience). 250 μL of the ENA medium prepared in (1)above were added to the wells seeded with epithelial tumor cellsfollowed by culturing at 37° C. and oxygen concentration of 1% (to bereferred to as “hypoxic culturing”) or oxygen concentration of 20% (tobe referred to as “normoxic culturing”). The medium was replaced everytwo days from the start of culturing. FIG. 3 depicts images indicatingcultured organoids on day 7 from the start of primary culturing (passage0).

Based on FIG. 3 , hypoxic culturing condition were confirmed to beessential for forming organoids in the case of the epithelial tumorcells used here.

In addition, FIG. 4 is a diagram indicating optimal culturing conditionsfor organoids established from 55 types of colon tumors. Based on FIG. 4, in the case of culturing normal colon epithelial organoids, incontrast to all constituents consisting of Wnt agonist composed of Wntprotein and R-spondin, EGF, p38 inhibitor, noggin and TGF-β inhibitorbeing required as constituents contained in the cell culture medium forculturing organoid, in the case of tumor cells, culturing was confirmedto be possible with fewer constituents.

However, there were cases in which a portion of the constituents (andespecially p38 inhibitor) exacerbated culturing efficiency accompanyingtumorigenesis (refer to the areas containing diagonal lines in FIG. 4 ).In addition, culturing efficiency worsened under 20% oxygen condition(normoxic condition), while on the other hand, there were cases in whichculturing was possible under 1% oxygen condition (hypoxic condition)(refer to the areas containing horizontal lines in FIG. 4 ). Here, coloncancer IV refers to metastatic colon cancer.

Consequently, all tumors were unable to be cultured under a single setof culturing conditions, and establishment efficiency was clearlydetermined to improve by preliminarily setting the eight culturingconditions indicated below. In the following culturing conditions, WR(−)indicates that Wnt agonist composed of Wnt protein and R-spondin is notcontained, while WR(+) indicates that Wnt agonist composed of Wntprotein and R-spondin is contained.

-   -   ENA/WR(−) medium, normoxic culturing    -   ENA/WR(+) medium, normoxic culturing    -   ENAS/WR(−) medium, normoxic culturing    -   ENAS/WR(+) medium, normoxic culturing    -   ENA/WR(−) medium, hypoxic culturing    -   ENA/WR(+) medium, hypoxic culturing    -   ENAS/WR(−) medium, hypoxic culturing    -   ENAS/WR(+) medium, hypoxic culturing

Example 3

(1) Preparation of Wnt3a-Afamin Complex

Wnt3a-afamin complex (to also be referred to as “W^(Afm)”) was preparedby utilizing the fact that bovine afamin is contained in fetal calfserum, culturing cells introduced only with Wnt3a gene inserum-containing medium, and culturing by utilizing the fact that thesecreted Wnt3a automatically forms a stable complex with afamin. Inother words, cells expressing Wnt3a having a tag sequence on theN-terminal thereof (W-Wnt3a/HEK) were cultured for 5 days to 7 days in adish or multilayered flask containing 10& fetal calf serum followed byrecovery of the culture supernatant. Continuing, the recovered culturesupernatant was subjected to centrifugal separation and passed through afilter (0.22 μm). 220 mL of the collected culture supernatant was used.Continuing, 200 mL of the recovered culture supernatant was added to 3mL of P20.1 antibody Sepharose, and after mixing by rotating for 3 hoursat 4° C., the medium was passed through an empty column to collect theSepharose. Furthermore, P20.1 antibody is an antibody that specificallyrecognizes the tag sequence on the N-terminal of Wnt3a. Continuing, theSepharose that collected in the column was washed with 3 mL ofTris-buffered saline (20 mM Tris-HCl, 150 mM NaCl, pH 7.5) and thiswashing procedure was repeated five times. Continuing, an eluent wasrecovered by eluting using 3 mL of peptide solution (0.2 mg/mL PAR4-C8peptide/TBS) per elution. This elution procedure was repeated ten timesto obtain Wnt3a-afamin complex. Wnt3a activity was confirmed by a TCFreported assay using W-Wnt3a/HEK cell supernatant and Wnt3a wasconfirmed to have a high level of activity in comparison withcommercially available Wnt3a (R&D Systems).

(2) Preparation of Cell Culture Medium for Organoid Culturing

First, human recombinant R-spondin 1 (R&D Systems) was added to acommercially available Advanced DMEM/F-12 medium (Thermo FisherScientific) to a final concentration of 1 μg/mL followed by the additionof noggin (Peprotech) to a final concentration 100 ng/mL and A83-01(Tocris) to a final concentration of 500 nM. Moreover, a medium wasprepared in which culture supernatant derived from W-Wnt3a/HEK culturedin serum-containing medium was added to a final concentration of Wnt3aof 300 ng/mL, IGF1 (Biolegend) was added to a final concentration of 100ng/mL, and FGF2 (Peprotech) was added to a final concentration of 50ng/mL.

In addition, human recombinant R-spondin 1 (R&D Systems) was added to acommercially available Advanced DMEM/F-12 medium (Thermo FisherScientific) to a final concentration of 1 μg/mL followed by the additionof noggin (Peprotech) to a final concentration 100 ng/mL and A83-01(Tocris) to a final concentration of 500 nM. Moreover, medium wasprepared in which the Wnt3a-afamin complex prepared in (1) above wasadded to a final concentration of 300 ng/mL, IGF1 (Biolegend) was addedto a final concentration of 100 ng/mL, and FGF2 (Peprotech) was added toa final concentration of 50 ng/mL (and the resulting medium is alsoreferred to as “W^(Afm)IFNRA medium”).

In addition, human recombinant R-spondin 1 (R&D Systems) was added to acommercially available Advanced DMEM/F-12 medium (Thermo FisherScientific) to a final concentration of 1 μg/mL followed by the additionof noggin (Peprotech) to a final concentration 100 ng/mL and A83-01(Tocris) to a final concentration of 500 nM as Comparative Example 1.Moreover, a medium was prepared in which culture supernatant derivedfrom W-Wnt3a/HEK culture in serum-containing medium was added to a finalconcentration of Wnt3a of 300 ng/mL, EGF (Thermo Fisher Scientific) wasadded to a final concentration of 50 ng/mL, and SB202190 (Sigma-Aldrich)was added to a final concentration of 10 μM (and the resulting medium isalso referred to as “WENRAS medium”).

In addition, human recombinant R-spondin 1 (R&D Systems) was added to acommercially available Advanced DMEM/F-12 medium (Thermo FisherScientific) to a final concentration of 1 μg/mL followed by the additionof noggin (Peprotech) to a final concentration 100 ng/mL and A83-01(Tocris) to a final concentration of 500 nM as Comparative Example 1.Moreover, a medium was prepared in which the Wnt3a-afamin complexprepared in (1) above was added to a final concentration of 300 ng/mL,EGF (Thermo Fisher Scientific) was added to a final concentration of 50ng/mL, and SBS02190 (Sigma-Aldrich) was added to a final concentrationof 10 μM (and the resulting medium may be referred to as “W^(afn)ENRASmedium”).

(3) Culturing of Intestinal Stem Cells

A lesion site from a colon tumor, or a portion at least 5 cm away fromthe colon tumor treated as normal mucosa, was sampled from colon tumorpatients provided with an explanation and from whom consent had beenobtained based on an ethics research protocol approved by the ethicscommittee of Keio University School of Medicine. Epithelial cells wereextracted from the sampled tissue with EDTA or Liberase TH followed byembedding in Matrigel®.

The Matrigel® containing the epithelial cells (to be referred to as“intestinal stem cells”) was seeded into a 48-well plate and culturedtogether with medium. More specifically, the procedure was as indicatedbelow.

The cultured intestinal stem cells were seeded into a 48-well platetogether with 25 μL of Matrigel® (BD Bioscience). 250 μl aliquots of thefour types of media prepared in (2) (“WIFNRA medium”, “W^(Afm)IFNRAmedium”, “WENRAS medium” and “W^(Afm)ENRAS medium”) were added to eachwell followed by culturing at 37° C. and oxygen concentration of 20%.The medium was replaced every two days from the start of culturing. FIG.5(A) shows images depicting the states of the cultures on day 7 from thestart of primary culturing (passage 0), while FIG. 5B is a graphquantifying the area in the wells occupied by organoids of intestinalstem cells in the media. In FIGS. 5(A) and 5(B), “W” indicates Wnt3acontaining serum while “W^(Afm)” indicates serum-free Wnt3a-afamincomplex.

Based on FIGS. 5(A) and 5(B), in the case of using “WENRAS medium”representing conventional culturing condition, the efficiency at whichorganoids were formed from single intestinal stem cells was poor and theorganoids did not grow to an adequate size. On the other hand, in thecase of using “WIFRNA” medium, organoids were able to be formed fromsingle intestinal stem cells with better efficiency. Moreover, in thecase of substituting conventional serum-containing Wnt3A with serum-freeWnt3a-afamin complex, the culturing efficiency of single normalepithelial cells with IGF1 and IGF2 improved dramatically.

Accordingly, the cell culture medium for culturing organoid of thepresent embodiment was clearly determined to be extremely effective forimproving the efficiency of forming organoids from single cells inapplications such as high-throughput screening of normal epithelialcells.

Example 4

(1) Preparation of Wnt3a-Afamin Complex

Wnt3a-afamin complex was prepared using the same method as in section(1) of Example 3.

(2) Preparation of Cell Culture Medium for Organoid Culturing

“WIFNRA medium”, “W^(Afm)IFNRA medium” and “WENRAS” medium were preparedusing the same method as in section (2) of Example 4. In addition,recombinant human R-spondin 1 (R&D Systems) was added to commerciallyavailable Advanced DMEM/F-12 medium (Thermo Fisher Scientific) to afinal concentration of 1 μg/mL followed by the addition noggin(Peprotech) to a final concentration of 100 ng/mL and A83-01 (Tocris) toa final concentration of 500 nM. Moreover, a medium was prepared inwhich the Wnt3a-afamin complex prepared in (1) above was added to afinal concentration 300 ng/mL, IGF1 (Biolegend) was added to a finalconcentration of 100 ng/mL, FGF2 (Peprotech) was added to a finalconcentration of 50 ng/mL, and SBS02190 (Sigma-Aldrich) was added to afinal concentration of 10 μM (and the resulting medium may be referredto as “W^(Afm)IFNRAS medium”).

(3) Culturing of Epithelial Tumor Cells Derived from Colon Tumor

Epithelial tumor cells were obtained using the same method as in section(2) of Example 1. Next, the epithelial tumor cells were seeded in a48-well plate together with 25 μL of Matrigel® (BD Bioscience). The“WIFNRA medium”, “W^(Afm)IFNRA medium”, “WENRAS medium” and “WAfmIFNRASmedium” prepared in (2) above were added in 250 μL aliquots to the wellsseeded with epithelial tumor cells followed by culturing at 37° C. andoxygen concentration of 20%. The medium was replaced every two days fromthe start of culturing. FIG. 6 depicts images indicating the states ofthe cultures six days after the start of the third round of subculturing(Passage 3) (total number of days cultured after start of culturing:26), six days after the start of the fourth round of subculturing(Passage 4) (total number of days cultured after start of culturing:33), and six days after the start of the sixth round of subculturing(Passage 6) (total number of days cultured after start of culturing:47). In FIG. 6 , “W” indicates Wnt3a containing serum while “W^(Afm)”indicates serum-free Wnt3a-afamin complex.

Based on FIG. 6 , the epithelia tumor cells derived from colon tumorused here exhibited toxicity to p38 inhibitor in the form of SB202190and did not grow in the presence of SBS202190 (“WENRAS medium” and“W^(Afm)IFNRAS medium”). On the other hand, organoid formation byepithelial tumor cells was observed as a result of culturing with mediumcontaining IGF1 and FGF2 (“WIFNRA medium”).

Moreover, Wnt agonist consisting of Wnt protein and R-spondin wasessential for the epithelial tumor cells derived from colon tumor usedhere, and during culturing with medium containing Wnt3a (“WIFNRAmedium”), growth was observed to be inhibited by serum contained inWnt3a and long-term culturing was not possible. On the other hand,long-term culturing was possible in the case of culturing with mediumcontaining serum-free Wnt3a-afamin complex (“W^(Afm)IFNRA medium”).

In addition, FIG. 7 is a table indicating the results of evaluatingorganoid formation efficiency and the possibility of long-term culturingin different compositions of the cell culture medium for culturingorganoid of the present embodiment. In FIG. 7 , ∘ indicates thatorganoids can be formed and that long-term culturing is possible forabout one month, ⊚ indicates that organoids can be cultured efficientlyand that long-term culturing is possible for about month, and ⊚⊚indicates that organoids can be formed even more efficiently and thatlong-term culturing is possible for about three months. In addition, inFIG. 7 , “Normoxia” refers to normoxic culturing condition at an oxygenconcentration of about 20%, while “Hypoxia” refers to hypoxic culturingcondition at an oxygen concentration of about 1%.

Based on FIG. 7 , according to the cell culture medium for culturingorganoid or culture method of the present embodiment, organoids wereshown to be able to be formed and long-term culturing was shown to bepossible, regardless of the cells or tissue, with respect to epithelialstem cells or epithelial tumor cells derived from mammals, includinghumans, tissue containing at least any one of these cells, or tissueunable to be cultured in the prior art.

According to the cell culture medium for culturing organoid of thepresent embodiment, epithelial stem cells, epithelial cells orepithelial tumor cells derived from mammals, including humans, tissue atleast containing any one of these cells, or tissue unable to be culturedin the prior art, can be cultured for a long period of time.

In addition, an organoid can be formed with high efficiency from atleast any one of the aforementioned cells and aforementioned tissue.

In addition, the differentiation ability of epithelial stem cellscultured using the cell culture medium for culturing organoid of thepresent embodiment can be maintained over a long period of time and thetumor incidence thereof is extremely low.

Moreover, an organoid obtained by the culture method of the presentembodiment can be used in regenerative medicine, basic medical researchon epithelial cells, drug response screening and drug developmentresearch using patient-derived epithelial organoids.

The invention claimed is:
 1. A method for producing an organoidcomprising: preparing an extracellular matrix, adhering epithelial stemcells, epithelial cells, epithelial tumor cells, or tissue at leastcontaining any one of these cells onto the extracellular matrix, andforming the organoid by adding a cell culture medium for culturing theorganoid and culturing the epithelial stem cells, the epithelial cells,the epithelial tumor cells, or tissue at least containing any one ofthese cells, after adhering, wherein the cell culture medium forculturing the organoid comprises at least two types of componentsselected from the group consisting of insulin-like growth factor 1(IGF1), fibroblast growth factor 2 (FGF2) and epiregulin (EREG), and atleast one type of component among the following components i) to iii):i) Wnt agonist, ii) bone morphogenetic protein (BMP) inhibitor, and iii)transforming growth factor-β (TGF-β) inhibitor, wherein the cell culturemedium for culturing the organoid does not substantially contain EGF andp38 inhibitor.
 2. The method for producing an organoid according toclaim 1, wherein the cell culture medium for culturing the organoidcontains IGF1 and FGF2.
 3. The method for producing an organoidaccording to claim 1, wherein the Wnt agonist is at least one typeselected from the group consisting of Wnt protein, R-spondin, and GSK-3βinhibitor.
 4. The method for producing an organoid according to claim 3,wherein the cell culture medium further comprises afamin and the Wntprotein forms a complex with the afamin, which stabilizes the Wntprotein.
 5. The method for producing an organoid according to claim 3,wherein the Wnt agonist is Wnt protein and R-spondin.
 6. The method forproducing an organoid according to claim 3, wherein the Wnt protein isat least one type selected from the group consisting of Wnt1, Wnt2,Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a,Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, and Wnt16.
 7. The method forproducing an organoid according to claim 3, wherein the R-spondin is atleast one type selected from the group consisting of R-spondin 1,R-spondin 2, R-spondin 3, and R-spondin
 4. 8. The method for producingan organoid according to claim 1, wherein the BMP inhibitor is at leastone type selected from the group consisting of noggin, gremlin, chordin,chordin-like protein containing a chordin domain, follistatin,follistatin-related protein containing a follistatin domain, DAN,DAN-like protein containing a DAN cysteine knot domain, sclerostin(SOST), and α-2 macroglobulin.
 9. The method for producing an organoidaccording to claim 1, wherein the BMP inhibitor is noggin.
 10. Themethod for producing an organoid according to claim 1, wherein the TGF-βinhibitor is at least one type selected from the group consisting ofA83-01, SB-431542, SB-505124, SB-525334, SD-208, LY-36494, and SJN-2511.11. The method for producing an organoid according to claim 1, whereinthe TGF-β inhibitor is A83-01.
 12. The method for producing an organoidaccording to claim 1, wherein the cell culture medium for culturing theorganoid is a serum-free medium.